Feline t-lymphotropic lentivirus

ABSTRACT

Compositions derived from a novel viral isolate designated feline T-lymphototropic lentivirus (FTLV) include the whole virus; proteins, polypeptides and, polynucleotide sequences derived from the virus; and antibodies to antigenic sites on the virus. These compositions are useful in a variety of techniques for the detection of and vaccination against FTLV. Detection methods disclosed include immunoassays for both the virus and antibodies to the virus, and the use of polynucleotide probes to detect the viral genome. Vaccines include both wholly and partially inactivated viruses and subunit vaccines. Whole, live virus is also useful as a model system for predicting the behavior of human immunodeficiency virus (HIV).

This invention was made with Government support under Grant No. CA 39016with the National Institutes of Health and the University of California.The Government has certain rights in this invention.

This is a division of application Ser. No. 07/089,700, filed Aug. 26,1987, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the detection and treatmentof viral infection. More particularly, the invention relates tocompositions and methods useful for the diagnosis of and vaccinationagainst infection with a newly-discovered lymphotropic retrovirus,designated feline T-lymphotropic lentivirus.

Domestic cats may become infected with several retroviruses, includingfeline leukemia virus (FeLV), feline sarcoma virus (FeSV), endogenoustype C oncornavirus (RD-114), and feline syncytia-forming virus (FeSFV).Of these, FeLV is the most significant pathogen, causing diversesymptoms, including lymphoreticular and myeloid neoplasms, anemias,immune-mediated disorders, and an immunodeficiency syndrome which issimilar to human acquired immune deficiency syndrome (AIDS). Recently, aparticular replication-defective FeLV mutant, designated FeLV-FAIDS, hasbeen more particularly associated with immunosupressive properties.

While immunodeficiency syndrome in cats has normally been associatedwith FeLV, immunodeficiency-like symptoms have been observed in catswhich are seronegative for FeLV, usually without alternativeexplanation. It would be desirable to identify etiological agents otherthan FeLV which are responsible for causing immunodeficiency in cats. Itwould be particularly desirable to provide methods and compositions forthe detection of and vaccination against such newly-identifiedetiological agents.

2. Description of the Background Art

The discovery of feline T-lymphotropic lentivirus was first reported inPedersen et al. (1987) Science 235:790-793. Abstracts concerning thediscovery of the virus have been presented at the American Associationfor Cancer Research on May 23, 1987 (Abstract No. 3337); and The ThirdInternational Conference on Acquired Immune Deficiency Syndrome, June1-5, 1987. A poster concerning discovery of the virus was presented at ameeting of the Federation of American Society for Experimental Biologyon April 2, 1987.

SUMMARY OF THE INVENTION

Compositions and methods are provided for detection of and vaccinationagainst a novel feline retrovirus designated feline T-lymphotropiclentivirus (FTLV). The compositions include the whole virus and portionsthereof, particularly including polypeptides which are cross-reactivewith antibodies specific for determinant sites characteristic of thevirus, such as those found on the major envelope and core proteins. Thecompositions further include antibodies capable of reacting with thevirus and polynucleotides which are capable of duplexing with the FTLVgenome. The FTLV virus will also serve as a useful model for othermammalian retroviruses, particularly the human immunodeficiency virus(HIV) responsible for acquired immunodeficiency syndrome (AIDS).

Using the compositions of the present invention, the virus and viralinfection may be detected by a variety of techniques, particularlyimmunoassays and techniques employing nucleotide probes. Immunoassaysprovide for the detection of the virus or antibody to the virus in aphysiological speciman, particularly blood and lymph tissue. Nucleotideprobes are used to detect the presence of the FTLV genome in aphysiological specimen. Vaccines may be prepared from the whole virus,either by partial or complete inactivation. Alternatively, subunitvaccines may be prepared from antigenic portions of the viral proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the Mg⁺² -dependent ( ) and Mn⁺² -dependent ( )reverse transcriptase (RT) activity and culture fluids containing FTLV,HIV, and FeLV. FeLV-producer line, FL-74, and HIV-infected H9 cells,were clarified by centrifugation at 3,000 rpm for two hours. Portions ofcell-free fluids (1 ml) were centrifuged at 17,000 rpm for one hour, andthe virus pellets were assayed for RT activity. Conditions of the assayare described in the Experimental Section hereinafter.

FIG. 2 is a transmission electron micrograph of a lymphocyte cultureinfected with FTLV. Extracellular viral particles surrounding aT-lymphocyte (center,×25,000) and a budding particle from a T-lymphocyte(left inset,×80,000). Mature virions were ellipsoid (120×150 nm) inshape with typical lentivirus-type nucleocapsids (right inset,×80,000).

FIG. 3 illustrates the sucrose density gradient banding of FTLV. Viruswas concentrated from T-lymphocyte culture supernatants by low speedcentrifugation to remove subcellular debris and ultracentrifugation topellet the virion particles. Pelleted virions were then layered on a10/50% (W/V) continuous sucrose gradient in tris-base (pH 7.4)containing 0.1M NaCl and 1 mM EDTA, and centrifuged in a Beckman SW41rotor for three hours at 36,000 rpm. Fractions (0.5 ml) were collectedfrom the bottom of the gradient and assayed for RT activity ( ), proteinconcentration (∇), and density (•). The purification procedure yielded 1milligram of FTLV per liter of T-lymphocyte culture.

FIG. 4 is Western blot of FTLV-infected cell lysats with lane 1 derivedfrom a serum sample from cat 2429, lane 2 derived from a serum sample ofan antibody-negative SPF cat, lane 3 derived from pooled serum samplesfrom HIV-positive humans, and lane 4 derived from pooled serum samplesfrom HIV-negative humans. The bands which appear in lane 1 indicate thepresence of proteins in FTLV which appear to correspond to the majorcore protein p24, gag precursor protein p55, and endonuclease proteinp32 of HIV, or cellular HLA-D12 p32.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

A novel virus designated feline T-lymphotropic lentivirus (FTLV) hasbeen discovered and isolated in substantially pure form. The virus isinfectious in cats, causing a wide variety of symptoms, includingabortion, alopecia, anemia, chronic rhinitis, conjunctivitis, diarrhea,emaciation, enteritis, gingivitis, hematochezia, neurologicabnormalities, periodontitis, and seborrheic dermititis. The course ofthe disease is usually fatal.

The etiology, pathogenesis, and morphology of FTLV closely resemblethose of human immunodeficiency virus (HIV) and simian T-lymphotropicvirus III (SAIDS), which cause acquired immunodeficiency syndrome inhumans and primates, respectively. FTLV does not appear to beantigenically related to HIV or to SAIDS, but rather appears to be aspecies-adapted lentivirus that has existed in cats for some time.Preliminary surveys conducted by the inventors herein indicate that FTLVinfection in cats may be widespread, possibly accounting for asignificant proportion of the immunodeficiency symptoms found in catswho are free from FTLV infection.

FTLV is a feline immunodeficiency virus characterized as a retrovirus,more specifically as a lentivirus, which is tropic for T-lymphocytes ofthe host which it infects. The virus is also characterized by horizontaltransmission, and may further be characterized by vertical transmissionin at least some cases.

It is expected that FTLV is polymorphic, and reference to FTLV in thepresent application is intended to encompass the entire FTLV family,including a variety of strains which share substantial amino acidsequence and nucleotide sequence homology and which are immunologicallyrelated. Substantial amino acid sequence homology means at least about75% homology, usually at least about 80% homology, and frequently 90%homology and above in at least some of the viral genes and proteins. Forexample, the env, gag, or pol regions may display the requisitehomology, while the genome as a whole does not. In such cases, so longas the viruses are immunologically related, the viruses will beconsidered to be FTLV within the ambit of the present invention.

By immunologically related it is meant that the various strains willdisplay substantial serologic cross-reactivity with the newly-discoveredstrain which has been deposited. Serologic cross-reactivity is definedas the ability of an antiserum or antibodies specific for the depositedFTLV strain to react with other FTLV strains as well as the depositedstrain. Usually, immunologically related strains will cross-react withantibodies specific for more than one epitopic site, usually more thanfive epitopic sites, and frequently ten or more epitopic sites.

Conveniently, FTLV strains may be identified by Western blot analysiswhere purified virus is disrupted with a suitable detergent, e.g.,sodium dodecyl sulfate, and separated on a slab gel by electrophoresis.The separated polypeptide bands are transferred from the gel tonitrocellulose filter paper and visualized with labelled antibody. Themolecular weights of the various resolved bands may then be determinedby comparison to known molecular weight standards. Substantialsimilarity between the Western blot analysis of an unidentified virusand that of a known FTLV virus indicates that the unknown virus islikely an FTLV virus.

FTLV encodes an RNA-dependent DNA polymerase (reverse transcriptase)which is Mg⁺² -dependent with maximal activity occurring at a Mg⁺²concentration of approximately 5 mM and pH of approximately 7.8. FTLVbands at a density of about 1.15 gcm³ in a continuous sucrose gradient.Western blotting of FTLV-infected cell lysate yields major bands atapproximately 22 to 26 kD, usually about 24 kD; 50 to 60 kD, usuallyabout 55 kD; and 28 to 36 kD, usually about 32 kD.

FTLV may be isolated from the sera of infected cats by conventionaltechniques. For example, peripheral blood lymphocytes (PBL) may beisolated from the blood of infected cats and placed in suitable culturemedia. The cultures are incubated, with normal PBL's being periodicallyintroduced to the culture in order to maintain its viability as theoriginal cells are killed by the virus. The infected cells should beplaced in fresh culture medium periodically, and the virus may berecovered from the supernatant of the cell culture by sucrose-gradientseparation, or other known separation techniques.

The FTLV may also be obtained from other specimens, particularly fromthe lymph tissues of infected animals. The lymph tissues are broken andthen suspended in culture medium, and the procedures described above arethen carried out.

Compositions according to the present invention include the whole virus,as well as portions of the virus. The whole virus may be maintained inin vitro culture, as described above, or may be viably frozen at atemperature at or below about -78° C. (solid CO₂ -dry ice), usually inthe presence of agents which promote amorphous, vitreous solidificationrather than crystallization. Suitable agents include glycerol anddimethylsulfoxide. Portions of the FTLV of particular interest includethe structural and regulatory proteins encoded by the FTLV genome,including the envelope and core proteins, and fragments thereof.

Polypeptides of the present invention will be either haptenic orantigenic, including at least six amino acids, usually at least nineamino acids, and more usually twelve or more amino acids foundcontiguously within one of the natural FTLV proteins. Polypeptides willgenerally correspond to at least one epitopic site which ischaracteristic of FTLV. By characteristic, it is meant that the epitopicsite will allow immunologic detection of the virus in a physiologicalsample with reasonable assurance. Usually, it will be desirable that theepitopic site be immunologically distinct from (i.e., not cross-reactivewith antibodies which recognize) viruses other than FTLV. In some cases,however, it may be desirable that the epitopic site be immunologicallysimilar to other viruses.

The FTLV polypeptides may be natural, i.e., including the entire FTLVprotein or fragments thereof isolated from a natural source, or may besynthetic. The natural polypeptides may be isolated from the whole viruswhich is obtained as described above by conventional techniques, such asaffinity chromatography. Conveniently, polyclonal or monoclonalantibodies obtained according to the present invention (as described inmore detail hereinbelow) may be used to prepare a suitable affinitycolumn by well-known techniques. Such techniques are taught, forexample, in Hudson and Hay, Practical Immunology, Blackwell ScientificPublications, Oxford, United Kingdom, 1980, Chapter 8.

Synthetic polypeptides which are immunologically cross-reactive with anatural FTLV protein may be produced by either of two generalapproaches. First, polypeptides having fewer than about 100 amino acids,more usually fewer than about 80 amino acids, and typically fewer thanabout 50 amino acids, may be synthesized by the well-known Merrifieldsolid-phase synthesis method where amino acids are sequentially added toa growing chain (Merrifield (1963) J. Am. Chem. Soc., 85:2149-2156).

The second and preferred method for synthesizing the polypeptides of thepresent invention involves the expression in cultured cells ofrecombinant DNA molecules encoding a desired portion of the FTLV genome.The portion of the FTLV genome may itself be natural or synthetic, withnatural genes obtainable from the isolated virus by conventionaltechniques. Of course, the genome of FTLV is RNA, and it will benecessary to transcribe the natural RNA into DNA by conventionaltechniques employing reverse transcriptase. Alternatively,polynucleotides may be synthesized by well-known techniques. Forexample, short single-stranded DNA fragments may be prepared by thephosphoramidite method described by Beaucage and Carruthers (1981), Tet.Letters 22:1859-1862. Double-stranded fragments may then be obtainedeither by synthesizing the complementary strand and then annealing thestrands together under appropriate conditions, or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

The natural or synthetic DNA fragments coding for the desired FTLVprotein or fragment may be incorporated in a DNA construct capable ofintroduction to and expression in in vitro cell culture. Usually, theDNA constructs will be suitable for replication in a unicellular host,such as yeast or bacteria. They may also be intended for introductionand integration within the genome of cultured mammalian or othereukaryotic cells. DNA constructs prepared for introduction into bacteriaor yeast will include a replication system recognized by the host, theFTLV DNA fragment encoding the desired polypeptide product,transcriptional and translational initiation regulatory sequences joinedto the 5'-end of the FTLV DNA fragment, and transcriptional andtranslational termination regulatory sequences joined to the 3'-end ofthe fragment. The transcriptional regulatory sequences will include aheterologus promoter which is recognized by the host. Conveniently, avariety of suitable expression vectors are commercially available for anumber of hosts.

To be useful in the detection methods of the present invention, thepolypeptides are obtained in a substantially pure form, that is,typically from about 50% W/W or more purity, substantially free ofinterfering proteins and contaminants. Preferably, the FTLV polypeptidesare isolated or synthesized in a purity of at least 80% W/W, and morepreferably, in at least about 95% W/W purity. Using conventional proteinpurification techniques, homogeneous polypeptide compositions of atleast about 99% W/W purity can be obtained. For example, the proteinsmay be purified by use of the antibodies described hereinafter using theimmunoabsorbant affinity columns described hereinabove.

Once a sufficient quantity of FTLV polypeptides have been obtained,polyclonal antibodies specific for FTLV may be produced by in vitro orin vivo techniques. In vitro techniques involve in vitro exposure oflymphocytes to the antigenic polypeptides, while in vivo techniquesrequire the injection of the polypeptides into a wide variety ofvertebrates. Suitable vertebrates are non-human, including mice, rats,rabbits, sheep, goats, and the like. Polypeptides having more than aboutthirty amino acids, usually more than about fifty amino acids, may servedirectly as the immunogen. If the polypeptide is smaller than about10kD, particularly less than about 6kD, however, it may be necessary tojoin the polypeptide to a larger molecule to elicit the desired immuneresponse. The immunogens are then injected into the animal according toa predetermined schedule, and the animals are bled periodically withsuccessive bleeds having improved titer and specificity. Injections maybe made intramuscularly, subcutaneously, or the like, and an adjuvent,such as incomplete Freund's adjuvent, will usually be employed. Thewhole virus can also be used as the immunogen, although selection ofantibodies specific for a particular determinant will be more difficult.

If desired, monoclonal antibodies can be obtained by preparingimmortalized cell lines capable of producing antibodies having thedesired specificity. Such immortalized cell lines may be produced in avariety of ways. Conveniently, a small vertebrate, such as a mouse, ishyperimmunized with the desired antigen by the method just described.The vertebrate is then killed, usually several days after the finalimmunization, the spleen removed, and the spleen cells immortalized. Themanner of immortalization is not critical. Presently, the most commontechnique is fusion with a myeloma cell fusion partner, as firstdescribed by Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519.Other techniques include EBV transformation, transformation withoncogenes, retroviruses, etc., or any other method which provides forstable maintenance of the cell line and production of monoclonalantibodies.

When employing fusion with a fusion partner, the manner of fusion is notcritical and various techniques may be employed. Conveniently, thespleen cells and myeloma cells are combined in the presence of anon-ionic detergent, usually polyethylene glycol, and other additivessuch as Dulbecco's Modified Eagle's medium, for a few minutes. At theend of the fusion, the non-ionic detergent is rapidly removed by washingthe cells. The fused cells are promptly dispensed in small culture wells(usually in a microtiter plate at relatively low density, ranging fromabout one to 5×10⁵ cells/well), in a selective medium chosen to supportgrowth of the hybrid cells while being lethal to the myeloma cells.Usually, the myeloma cell line has been mutated to be sensitive to alethal agent, typically being HAT sensitive, and the medium includes aHAT concentration sufficient to inhibit the proliferation of the unfusedmyeloma cells.

After sufficient time, usually from about one to two weeks, colonies ofhybrids are observed and plates containing hyperpositive wells areidentified. The plates and wells having only one colony per well areselected, and supernatants from these wells are tested for bindingactivity against FTLV or a particular FTLV protein. Once positivehybridomas are identified, the cell line can be maintained as a viableculture and/or a quantity of the virus may be grown out, separated, andstored by lyophilization.

Depending on the desired use for the antibodies, further screening ofthe hybridomas may be desirable. For use in immunodiagnostic assays,antibodies having very high specificity and affinity for the antigenicsite are desirable.

Once the desired hybridomas have been selected, monoclonal antibodiesmay be isolated from supernatants of the growing colonies. The yield ofantibodies obtained however, is usually low. The yield may be enhancedby various techniques, such as injection of the hybridoma cell line intothe peritoneal cavity of a vertebrate host. Monoclonal antibodies maythen be harvested from the ascites fluid or the blood. Proteinaceous andother contaminants will usually be removed from the monoclonalantibodies prior to use by conventional techniques, e.g.,chromatography, gel filtration, precipitation, extraction, or the like.

The polypeptides and antibodies of the present invention may be usedwith or without modification for the detection of or vaccination againstFTLV infection. Frequently, the polypeptides and antibodies will belabelled by joining, either covalently or non-covalently, a substancewhich provides for detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Some of the labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers,chemiluminescers, magnetic particles and the like. Patents teaching theuse of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Antibodies and polypeptides prepared as described above can be used invarious immunological techniques for detecting FTLV and anti-FTLVantibodies in physiological specimens, particularly body fluid samples,including blood, plasma, serum, urine, and the like, and cell samples,such as lymphocytes. Depending on the nature of the sample, bothimmunoassays and immunohistochemical staining techniques may find use.

Liquid phase immunoassays and Western blot analysis will find use indetection of FTLV in body fluids, particularly blood and urine. The useof antibodies in protein binding assays is well established. Numerouscompetitive and noncompetitive protein binding assays have beendescribed in the scientific and patent literature, and a large number ofsuch assays are commercially available. Detailed methods for detectingthe presence of the viruses in serum samples are set forth in theExperimental section hereinafter. Additionally, enzyme linkedimmunosorbent assays (ELISA) for detecting presence of antibodies toFTLV in blood are also set forth in the Experimental section.

Compositions of the present invention are also useful in preparingvaccines for protection against FTLV infection. For example, the wholevirus may be wholly or partially inactivated. Partial inactivation maybe achieved by passage at elevated temperatures or by contact withmutagens, such as ultraviolet light, ethyl methanesulfonate, and thelike. Complete inactivation may be achieved by contact with otheragents, including formalin, phenol, α-lactopropianate, ultravioletlight, heat, psorlens, platinum complexes, ozone and other viricidalagents.

The viral proteins and portions thereof, prepared as described above,may also be used in the preparation of subunit vaccines prepared byknown techniques. Polypeptides displaying antigenic regions capable ofeliciting protective immune response are selected and incorporated in anappropriate carrier. Alternatively, an antigenic portion of a viralprotein or proteins may be incorporated into a larger protein byexpression of fused proteins. The preparation of subunit vaccines forother viruses is described in various references, including Lerner etal. (1981) Proc. Natl. Acad. Sci. USA 78:3403 and Bhatanagar et al.(1982) Proc. Natl. Acad. Sci. USA 79:4400. See also, U.S. Pat. Nos.4,565,697 (where a naturally-derived viral protein is incorporated intoa vaccine composition); 4,528,217 and 4,575,495 (where syntheticpeptides forming a portion of a viral protein are incorporated into avaccine composition). Other methods for forming vaccines employing onlya portion of the viral proteins are described in U.S. Pat. Nos.4,552,757; 4,552,758; and 4,593,002. The relevant portions of each ofthese cited references and patents are incorporated herein by reference.

The vaccines prepared as described above may be administered in anyconventional manner, including oronasally, subcutaneously, orintramuscularly, except that oronasal administration will usually not beemployed with a partially inactivated virus vaccine. Adjuvants will alsofind use with subcutaneous and intramuscular injection of completelyinactivated vaccines to enhance the immune response.

Diagnostic tests for detecting the presence of FTLV in biologicalsamples may also be performed using polynucleotide probes. Suchpolynucleotide probes may be prepared based on the sequence of the viralgenome. The length of the probe is not critical, but will usuallycomprise at least about 12 bases, more usually comprising at least about16 bases, which are substantially complementary to a portion of theviral genome. The probe itself may be DNA or RNA, and the probe need nothave perfect complementarity with the FTLV genome, with one or twomismatched pairs being acceptable for probes up to 20 bases in lengthand three to five mismatched pairs in probes from 20 to 35 bases. Theprobes may be prepared synthetically, with suitable synthetic techniqueshaving been described above, and will include a detectable label.Usually, the synthetic sequences are expanded in commonly availablecloning vectors and suitable hosts in order to obtain large quantities.The expanded vectors may themselves be labelled for use as probes, orshorter fragments containing complementary strands may be excised andlabelled. Methods for the preparation and utilization of nucleotideprobes for diagnostic testing are described in U.S. Pat. No. 4,358,535to Falkow et al., the disclosure of which is incorporated herein byreference.

A variety of labels have been employed, including those which have beendescribed above for use in immunoassays, particularly radionuclides.Suitable labels may be bound to the probe by a variety of techniques.Commonly employed is nick translation with α-³² P-dNTP terminalphosphate hydrolysis with alkaline phosphatase followed by 5'-endlabelling with radioactive³² P employing λ-³² P-NTP and T4polynucleotide kinase or 3'-end labelling with an α-³² P-dNTP andterminal deoxynucleotidyl transferase. Alternatively, nucleotides can besynthesized where one or more of the atoms present are replaced with aradioactive isotope, e.g., hydrogen with tritium. In addition, variouslinking groups can be employed. The terminal hydroxyl can be esterifiedwith inorganic acids, e.g., ³² P phosphate or ¹⁴ C organic acids, orelse esterified with bifunctional reagents to provide other reactivegroups to which labels can be linked.

The following examples are offered by way of illustration, not by way oflimitation.

EXPERIMENTAL Materials and Methods 1. Isolation and Culturing of FTLV

Peripheral blood lymphocytes (PBL) were isolated by Ficoll-Hypaquemethod (Boyam (1968) Scand. J. Clin. Lab. Invest. 97 (Suppl. 21):77)from 3-5 ml of heparinized blood of cats suspected of FTLV infection.The PBLs were resuspended in culture media at a final concentration of0.5-1.0×10⁶ cells/ml and placed in 25-cm² culture flasks. To this cellsuspension, mitogen-stimulated normal donor PBLs were added at normal toinfected PBL ratios of 1:1 or 1:2. Culture media consisted of RPMI 1640with 10% heat-inactivated fetal calf serum, 10 mM HEPES, 100 U/mlpenicillin, 10 μg/ml streptomycin, 50 μg/ml gentamycin, 2 mML-glutamine, 5×10⁻⁵ M 2-mercaptoethanol, 2 μg/ml polybrene, and 100 U/mlhuman interleukin-2(IL-2). Cultures were incubated in 5% CO₂ at 37° C.and monitored routinely (every other day) for syncytia formation andother cytopathic effects (i.e., cell death). Culture supernatants wereharvested for reverse transcriptase (RT) activity and cells wereresuspended in fresh culture media on every 3rd day or twice a week.These cultures were further supplemented with stimulated normal PBLsfrom the same donor to maintain minimum cell concentration of0.5-1.0×10⁶ cells/ml. Such a procedure detected FTLV at as early as Day5 and as late as Day 60 of the incubation period.

Virus production was monitored by syncytia formation and by reversetranscriptase (RT) assay using M_(g) ⁺⁺ as divalent cation,poly(rA)-oligo(dT₁₂₋₁₈) as template primer, and 5 μCi minimum of [3H]TTPper sample (Colche and Schlorn (1980) Biochim. Biophys. Acta 607:445).Cultures with positive RT activities (two consecutive harvests with RTactivity greater than 6,000 cpm/ml) were tested for the presence of FTLVantigen(s) on the cell surface or in the cells by immunofluorescenceassay (IFA) with pooled FTLV-positive serum and for the presence of FTLVvirions by electron microscopy. The cultures were considered positivefor FTLV isolation when results from above assays all concurred in thepresence of a feline lentivirus. The virus isolates from such cultureswere massively produced, purified by sucrose-gradient, and testedagainst FTLV isolate standard (Petaluma isolate) by Western blotanalysis using pooled FTLV-positive serum. Those isolates showing one ormore bandings comparable to FTLV standard were then considered FTLVisolates based on this antigenic comparison.

2. Isolation of FTLV From Other Specimen Samples

The procedures for specimen samples that are not PBLs were as follows.Cells from tissue specimen were teased and/or minced aseptically withforceps and scalpels, resuspended in culture media, and the proceduresdescribed for PBL samples were thereafter followed. Samples consistingof 0.5-1.0 ml of body fluids (i.e., serum, plasma, CNS fluid, saliva)were added to 5-ml cultures of stimulated donor PBL which was at 1×10⁶cells/ml, and the procedures described for PBL samples were thereafterfollowed.

3. Preparation of Stimulated PBLs From Normal Cat Donors

Specific-pathogen-free kittens and cats were used as normal donors ofPBLs. Donor PBLs were prepared from heparinized blood (5-40 ml) byFicoll-Hypaque method. The cells were resuspended in culture media (notsupplemented with polybrene or human IL-2) and cultured in the presenceof 5 μg/ml of concanavalin-A (Con A) in 5% CO₂ at 37° C. for three days.The stimulated donor PBLs were then harvested, washed with sterile 1×Hank's balanced salt solution, and resuspended in complete culture mediaready to be used for FTLV isolation, or resuspended in culture mediafree of polybrene and recultured for later use. Stimulated PBLs could begrown for at least three months and used for FTLV isolation without lossin virus recovery. Other mitogens were successfully used to stimulatefeline donor PBL for FTLV isolation, including Staphylococcalenterotoxin A (0.05 μg/ml), phytohemagglutinin (20 μg/ml),Staphylococcal protein A (5 μg/ml), and lipopolysaccharide (100 μg/ml).

4. Western Blot Analysis

A modification of the Western blot technique described by Carlson et al.(1985) J. Am. Med. Assn. 253:3405 was used. Sucrose-gradient purifiedvirus was disrupted with 0.1% SDS and electrophoresed on a 8%preparative polyacrylamide slab gel (with 5% stacking gel) in presenceof 0.1% sodium dodecyl sulfate (SDS). The separated viral proteins weretransferred electrophoretically onto a nitrocellulose filter which wasthen treated with Buffer 3 containing 3% gelatin and cut into strips ofidentical size. Two lanes of each gel containing molecular weightstandards were stained with amido black and later used for molecularweight comparison of the immunoblots.

Serum samples were diluted to 1:100 in Buffer 3 and incubated with blotstrips in separate glass tubes for 60 min. at 37° C. The blot stripswere washed individually and incubated with peroxidase-conjugated rabbitanti-cat IgG (Cappel Laboratories) at a dilution of 1:400 for 60 min. at37° C. After extensive washing, each strip was incubated with a freshsubstrate solution (0.05% diaminobenzidine and 0.01% H₂ O₂ in 0.1M Tris,pH 7.4) for 4-10 min. at room temperature. The reaction was stopped withexcess distilled H₂ O upon establishment of visible bands, and thestrips were blot dried. The molecular weights of the bands on theimmunoblots were then determined by comparing the migration distancewith molecular standards from the original gel. Positive and negativecontrol sera were included in each Western blot analysis as internalcontrol for diagnostic evaluation.

5. Indirect Immunofluoresence Assay (IFA) for Detecting FTLV Antibodiesand FTLV-Infected Cells

Concanavalin A-simulated normal cat PBLs at a concentration of 2×10⁶cells/ml in culture media were infected with FTLV at RT levels of5,000-10,000 cpm/ml and incubated in 5% CO₂ at 37° C. The culture mediaused throughout these studies consisted of RPMI-1640 media containing100 U/ml rIL-2, 2 fg/ml polybrene, 10% heat-inactivated FCS, 1×penicillin/streptomycin, 50 μg/ml gentamycin, 5×10⁵ M 2-mercaptoethanol,and 10 mM Hepes. The virus stocks were infected tissue culture fluid(TCF) (greater than 100,000 cpm/ml in RT activity) which were filtered(0.45 μm), aliquoted, and stored frozen at -70° C. The culture fluidswere harvested every 3-4 days, and the infected cells were recultured ina fresh culture media and supplemented with stimulated normal cat PBL tomaintain the live density of 2×10⁶ cells/ml. Virus from the infected TCFwas purified by sucrose gradient method (Pedersen et al. (1987) Science235:790), and used as antigens for ELISA and Western blot. Equal numberof infected PBLs from Day 3, 6, 9, 12, and long-term cultures from abovewere pooled, washed with phosphate buffered saline (PBS), air-dried on10-well IFA slides at 5×10⁶ cells/ml, acetone-fixed for 10 min andstored at -5° C. in a dessicator. These slides were thawed to roomtemperature just prior to IFA testing. Fifteen μl of 1:10 diluted serawere placed on individual wells and incubated for additional 30 min at37° C. with fluorescein-conjugated anti-cat IgG (AntibodiesIncorporated, Davis, Calif.) at a dilution of 1:75. After washing, theslides were counter-stained with 0.01% evans blue, mounted in bufferedglycerol, and read by fluorescent microscopy. The serum samples wereconsidered positive for FTLV antibodies when both giant cells andinfected cells gave typical membrane and cytoplasmic fluorescence.Positive and negative control serum were included in every assay.

6. Enzyme-Linked Immunosorbent Assay

(ELISA) for Detecting FTLV Antibodies

ELISA used in this study was a modification of the methods described byLutz et al. (1980) Cancer Res. 40:364 for FeLV and Carlson et al. (1985)supra for HIV. Sodium dodecylsulfate (0.1% SDS)-disrupted purified FTLVwere diluted in coating buffer (0.1M NaHCO₃, pH 9.6) to 2 μg/ml, and 0.1ml (200 ng) of this viral suspension was added to each well of theImmulon II microtiter plates. The plates were incubated for 12 to 16hrs. at 37° C., and then washed with wash solution (0.05%-Tween-20,0.15M NaCl). The test sera were diluted 1:100 in Buffer 3 (0.15M NaCl, 1mM edetic acid, 0.05% Tween-20, 0.1% bovine serum albumin, pH 7.4), and0.1 ml of the diluted sera were added to each well in duplicate andincubated for 60 min. at 37° C. The plates were then washed andincubated with 1:100 dilution of peroxidase-conjugated rabbit anti-catIgG (Cappel Laboratories, Conchranville, Penna.) for 30 min at 37° C.After another wash cycle, the plates were incubated with a freshsubstrate solution (50 mM citric acid adjusted to pH 4.0, 0.2 mM 2,2'-azinobis-3-ethylbenzthiazoline-sulfonic acid, 2 mM H₂ O₂) for 10 min.at room temperature. The reaction was stopped with the addition of 0.1ml of 0.2M hydrogen fluoride and the absorbance at 405 nm was measuredby an ELISA plate reader. Each ELISA plate contained duplicate wells ofpositive and negative control sera which were used throughout the studyas internal standards for diagnostic comparison.

7. Preparation of Inactivated Purified FTLV as a Whole Virus VaccineAgainst FTLV

Tissue culture fluid (TCF) infected with FTLV was centrifuged at 3,000rpm for 1 hr. to remove cellular debris, and the cell-free TCF waspelleted at 17,000 rpm for 2 hrs. The pelleted virus was then layered ona 10/50% (W/V) continuous sucrose gradient in TEN buffer tris-base(pH7.4) containing 0.1M NaCl and 1 mM EDTA and ultracentrifuged in aBeckman SW41 rotor for 3 hrs. at 36,000 rpm (Pedersen et al. (1987)supra). Peak viral fractions of the gradient were determined by thepresence of reverse transcriptase activity, and the peak fractionspooled, diluted with TEN buffer, and pelleted out of the sucrosesolution at 17,000 rpm for 2 hrs. The resulting purified virus wasinactivated by psoralen (Marx et al. (1986) J. Virol. 60:431) and thenby 0.8% formaldehyde at 4° C. for 24 hrs. (Swansstrom et al. (1981)Virology 113:613). The inactivated virus was pelleted out of theformaldehyde solution, resuspended in saline solution, and mixed withadjuvant (N-acetylmuramyl-L-alanine-D-isoglutamine, MDP) beforeinoculation.

RESULTS 1. Initial Isolation and Serial Transmission of FTLV

FTLV was isolated from a cattery of 43 cats which were all seronegativefor FeLV. A number of cats in one pen had died, and several had animmunodeficiency-like syndrome. Only one of 18 symptom free cats in thecattery showed serologic evidence of FTLV infection (by IFA), while tenof 25 cats showing signs of ill health were seropositive for the virusby IFA. The disease course and clinical signs observed in these ten cats(which died over a four-year-period) are set forth in Table 1.

                  TABLE I                                                         ______________________________________                                                 Duration                                                             Cat      of                                                                   Name     Illness    Clinical Signs                                            ______________________________________                                        Cv       3 years    Chronic rhinitis, conjunctivitis,                                             diarrhea, abortion, vague                                                     neurologic abnormalities,                                                     periodontitis, gingivitis,                                                    anemia, emaciation                                        GK       7 months   Chronic rhinitis, diarrhea,                                                   perodontitis, gingivitis, anemia,                                             emaciation                                                FL.sup.1 9 months   Diarrhea, hematochezia,                                                       periodontitis, gingivitis,                                                    alopecia, seborrheic dermatitis,                                              emaciation                                                SA.sup.1 7 months   Found extremely dehydrated,                                                   depressed and near death.                                                     Responded well to treatment but                                               fell into a pattern of                                                        depression, dehydration and                                                   weight loss. Severe diarrhea for                                              the last 2 weeks of life.                                 TC.sup.1 6 months   Found near death and died 5 hours                                             later. Appeared healthy the                                                   night before. History of                                                      periodontitis and gingivitis for                                              several months prior to death.                                                Evidence of severe chronic and                                                acute enteritis at necropsy.                              CY       hours      Found dead. Appeared healthy the                                              night before. Possible acute                                                  enteritis.                                                RU       hours      Found dead. Appeared healthy the                                              night before. Possible acute                                                  enteritis.                                                BL       1 month    Chronic diarrhea, anorexia,                                                   dehydration, emaciation.                                  CH.sup.2 1 month    Thin, seborrheic dermatitis,                                                  chronic rhinitis, anemia.                                 CL       5 months   Found depressed and hypothermic                                               with terminal hysteria and rage.                          ______________________________________                                         .sup.1 The new retrovirus was isolated from blood of cat FL and from          pooled plasma from cats SA and TC.                                            .sup.2 Tested positive for FTLV BY IFA prior to death. All other cats die     before testing for FTLV.                                                 

An FTLV strain designated feline T-lymphototropic lentivirus, Petalumaisolate, was deposited at the american Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, on Aug. 5, 1987 and grantedAccession No. VR2186.

Given the possible infectious nature of the disease syndrome, an attemptwas made to serially transmit the disease from affected cattery cats tospecific pathogen-free (SPF) kittens. Whole blood (1 ml) from cat FL wasinoculated intraperitoneally into SPF kitten 2428. A second SPF kitten(2429) was inoculated intraperitoneally with 2 ml of filtered (0.22 fM)pooled plasma from cats SA and TC. Plasma from these donor cats was usedinstead of blood because both of them tested positive for FeSFVinfection. FeSFV is highly cell-associated and very little virus occursfree in plasma. Plasma buffy coat preparations, and bone marrow samplesfrom cats FL and SA were also cocultivated with Fc9, Fcwf-4, and Crfkfeline fibroblasts. Cultures were monitored for cytopathic effect (CPE),Mg⁺² - and Mn⁻² -dependent reverse transcriptase (RT) activity, andFeLV-p27 antigen expression by ELISA. The cocultures with plasma andbuffy coat cells were discarded after they were negative for 6 weeks.Bone marrow cultures from cat SA yielded FeSFV after several passagesbut were negative when examined by transmission electron microscopy forother agents.

Kittens 2428 and 2429 were monitored daily for clinical signs of illnessand complete blood counts were taken weekly. A generalized enlargementof peripheral lymph nodes was noticed beginning 4 weeks afterinoculation. A low-grade fever appeared 2 weeks later and was associatedwith a precipitous drop in the total leukocyte count and absolutenumbers of polymorphonuclear neutrophils. Platelet and red blood cellcounts remained normal, however. Cat 2429 developed a severe, bacterialcellulitis at the site of ear tagging, septicemia, and high fever withina week of the time that the white cell count decreased. The infectionand high fever disappeared after broad spectrum antibiotic therapy wasinstituted. The leukopenia and low-grade fever disappeared after 2 to 4weeks, but the generalized lymphadenopathy persisted for at least 5months after inoculation.

Buffy coats were prepared from blood samples collected from the twoexperimentally inoculated kittens (2428 and 2429) during the leukopenicphase of their illness. The buffy coats were cocultured with stimulatedperipheral blood lymphocytes (PBL) from normal SPF cat donors. The donorlymphocytes were harvested from fresh whole blood by means ofFicoll-Hypaque gradients and were stimulated with concanavalin A (Con A;5 μg,/ml). Three days after Con A stimulation, nonadherent bloodleukocytes were transferred to fresh flasks and further stimulated withrecombinant human interleukin-2 (IL-2; 100 U/ml). Cells examined after 7days in culture were more than 95% T lymphocytes as determined by an IFAwith the use of rabbit antiserum to feline theta, immunoglobulin G(IgG), and immunoglobulin M (IgM) antigens. Cultures were replenishedwith fresh Con A and IL-2-stimulated PBL every 5 to 7 days. A cytopathiceffect (CPE) consisting of ballooning degeneration, increased celldeath, and giant cell formation was noted in cultures of cells from cat2429 within 14 to 21 days of cocultivation. A similar CPE was seen incultures from cat 2428 after 4 to 6 weeks. Cytopathic changes wereassociated with the appearance of RT activity that was totally Mg⁻²-dependent. The RT activity of FTLV resembled that of the HIV anddiffered from that of FeLV (FIG. 1). Lymphoid cell cultures showing CPEand RT activity were negative for FeLV-p27 antigen by ELISA and forFeSFV by IFA. Transmission electron micrographs of RT and CPE-positivecultures revealed mature, immature, and budding particles typical oflentiviruses (FIG. 2). The particles were slightly smaller and ellipsoidthan HIV and had more prominent envelope spikes. Particles with themorphology of type C or D oncornaviruses or FeSFV were not observed.Virus purified from feline T-lymphoid cultures banded at a density of1.15 g/cm³ in continuous sucrose gradients (FIG. 3).

Reverse transcriptase levels in culture supernatants increasedprogressively after each serial passage of the virus in fresh PBL andpeaked after day 40 to 45. After exposure of fresh PBL with infectedcell-free culture supernatants, the RT level in the culture increasedprogressively and peaked by day 7. Infectivity for T-lymphocyte enrichedPBL could be readily demonstrated with both filtered tissue culturefluid and cellular inocula. The virus readily infected FL-74 and LSA-Icell lines, both of which are feline T-lymphotropic cell lineschronically infected with FeLV. The virus failed to replicate, however,in several feline, fibroblastic cell lines, including Fc9, Fcwf-4, andCrfk. Infectivity studies with long-term human T-lymphoid cell lines(H9, HUT 78) and with primary cultures of human PBL stimulated withphytohemagglutinin and IL-2 have been negative.

The serologic relation between FTLV and HIV was examined. Serum samplesfrom all 43 cats were uniformly negative against HIV when tested by IFAand Western blotting. Western blots, prepared from gradient-purifiedvirus from cat 2429 or from virus-infected cell lysates, reacted withsera from experimentally infected cat 2429 and with several cats fromthe cattery, but not with pooled human sera positive for HIV. Threehumans who had persistent and close contact with cats in the cattery hadno antibodies to HIV or to the new feline virus by IFA and Westernblotting.

A serologic survey of the cattery was conducted by using an IFA withinfected feline lymphocytes as the substrate. Most of the infectedanimals were confined to one pen, with only two infected animals foundin other pens. A thorough physical examination of all 43 cats in thecattery showed that 18 were healthy and 25 had various ailments.Unhealthy cats were either very thin and rough-coated or had one or moreof a number of chronic infections including gingivitis, periodontitis,pustular dermatitis, ear infections, chronic rhinitis, chronicconjunctivitis, or diarrhea. Only 1 of the 18 (5.6%) healthy cats wasseropositive for the new virus, whereas 10 of the 25 (40%) unhealthycats had antibodies to the new virus. None of the cats were infectedwith FeLV; however, several were seropositive for FeSFV and felineinfectious peritonitis virus (FIPV).

Clinical signs observed in the ten cats that were seropositive for thenew virus included chronic rhinitis, excessive thinness, and anemia. Oneof these animals had a recurrent bacterial cystitis that is uncommon incats. Periodontitis, stomatitis, gingivitis, miliary pustulardermatitis, bacterial otitis exema, and aural hematomas were recognizedin both seropositive and seronegative animals. Neurologic abnormalitieswere not observed in any of the surviving seronegative or seropositivecats; they were recognized, however, in two cats that had died prior tothe institution of testing for the new virus (Table 1).

A disease identical to that transmitted to SPF kittens 2428 and 2429inoculated with whole blood or plasma was subsequently transmitted toother SPF kittens by means of purified virus that had been propagated intissue culture. The cultured virus was then reisolated from the blood ofthese kittens several weeks later. An identical virus was alsorepeatedly reisolated from cats 2428 and 2429 over a period of 4 monthsor more, thus indicating the persistence of the infection. A limitedserologic survey of cats has confirmed the existence of the new virus indiseased cats from many different geographic areas of NorthernCalifornia, as well as in other states. Seropositive cats have also beenidentified in Canada and Japan.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. Feline T-lymphotropic lentivirus (FTLV) isolatedfrom cells grown in in vitro cell culture.
 2. FTLV as in claim 1,characterized by the Western blot of FIG. 4, lane
 1. 3. A biologicallypure culture of Feline T-lymphotropic virus (FTLV), strain Petaluma,A.T.C.C. Accession No. VR2186.